The decussation of the retinal ganglion cell (RGC) projections in the optic chiasm is essential for normal mapping of visual information, and insures that each hemisphere receives information from both eyes to establish binocular vision. How the development of the binocular pathway is determined has been a longstanding enigma. Recent work in the spinal cord has shown that distinct subpopulations of neurons express combinations of homeodomain transcription factors and that such code determines projection to specific targets. We hypothesized that a similar code of regulatory gene expression might designate the uncrossed and crossed subpopulations of RGCs. Our laboratory has discovered that a zinc-finger containing transcription factor Zic2, is expressed in RGCs with an uncrossed trajectory. Zic2, but not other Zic family members, is postmitotically but dynamically expressed in RGCs in ventrotemporal (VT) retina in the period when the uncrossed RGC subpopulation projects axons from this location toward the chiasm. Gain- and loss-of-function experiments in vitro indicate that Zic2 is necessary and sufficient to switch the outgrowth behavior of retinal axons when co-cultured with chiasm cells, from crossed to uncrossed patterns, or vice versa. Moreover, Zic2 expression correlates with the degree of binocular vision across several species. Other work in our lab has identified EphB1 as a receptor system interacting with the inhibitory factor ephrinB2 at the chiasm midline in mouse. EphB1 is expressed coincident with Zic2, spatially and temporally. The proposed work will further analyze the hypothesis that Zic2 specifies the uncrossed retinal ganglion cell axon trajectory, by regulating expression of guidance receptors in the Eph family that mediate retinal axon divergence at the optic chiasm midline. Aims include verification that Zic2 is expressed in ganglion cells with an uncrossed trajectory and elucidation of its temporal expression (Aim1); confirmation that Zic2 is necessary and sufficient to establish an uncrossed trajectory, by using loss- and gain-of-function strategies in vivo (Aim 2); investigation of Zic2's influence on genes that are putative targets, especially with regard to the EphB1 receptor in the retina (Aim 3). This analysis should shed light on patterning the binocular pathway and should apply to the establishment of bilateral sensory pathways elsewhere in the nervous system.